A primary focus of this laboratory is to elucidate the forces important for folding and recognition reactions of biological macromolecules. The direct measurement of forces between many biomolecules in macroscopic condensed arrays shows that at close separation forces seem to be dominated by the structuring of the intervening water. Work is progressing both on condensed arrays and on the binding reactions of DNA and proteins in dilute solution. Differences in the water sequestered between specific and nonspecific complexes can be determined from the dependence of the relative binding constants on water activity. In contrast to specific complexes, a full hydration layer between the apposing protein and DNA surfaces is retained in nonspecific complexes. The difference in the number of waters sequestered between nonspecific and specific complexes of EcoRI is now seen as temperature dependent. The dependence of this number of waters on temperature seems to correlate with the changes in the free energies of binding itself. At the very least, these changes in binding free energy are directly due to changes in the energy of water hydrating the two surfaces. Experiments are underway to measure the temperature dependence of pressure-volume work necessary to remove water from the nonspecific complex of EcoRI and DNA sequence that differs from the recognition sequence by one base pair. The ability to measure the work necessary to remove water from nonspecific complexes using relative binding constants is limited to comparatively low osmotic pressures since the kinetics become too slow for equilibrium measurement. We have now shown that the off-rates, however, contain all of the osmotic stress dependence of the equilibrium. Much higher pressures can now be applied and larger osmotic work determined. It is now possible to distinguish a two-state model for the loss of water from a nonspecific complex and a gradual loss using the kinetic measurements. A second interest of the laboratory is in using transient electric birefringence to characterize the flexibilities of macromolecular assemblies. We are measuring the Mg and ATP dependent flexible to stiff transition of the myosin rod that correlates with activity. Using genetically engineered myosin rods, the flexible conformation is seen as a locally melted coiled-coil localized at the HMM-LMM junction. Experiments are planned to probe the structural head junction coupling through the now predicted rotation accompanying the flexible-stiff transition.